PRKACA confers resistance to anti-HER2 therapy and impairs apoptosis. A. Relative viability of screened BT474 cells containing each ORF and treated with AEE788 (top panel) or an shRNA targeting HER2 (bottom panel). B. PRKACA confers resistance to lapatinib. Cells were lentivirally-transduced with the indicated ORFs, treated with lapatinib at the indicated doses, and cell viability was assessed by ATP-based luminescence assay. Results are normalized to the DMSO control for each ORF and represent the mean and standard deviation (SD) of 6 replicates. C. PRKACA expression prevents lapatinib-induced cell death. BT474 cells were transduced with LACZ or PRKACA constructs and subsequently treated with lapatinib 5 uM or DMSO. Viable cells were counted by trypan blue exclusion 5 days after the addition of drug. Results show the mean and standard deviation of 3 replicates. D. Lapatinib treatment results in apoptosis of BT474 (top panel) and ZR-75-30 (bottom panel) cells, and this is partially rescued by PRKACA, PIM1, and BCL-XL. Cells were transduced with the indicated ORFs. Two days later lapatinib 10 uM or DMSO was added, and caspase 3/7 cleavage was measured 24h later by luminescent assay. Results represent the mean and SD of 5 replicates per cell line. For BT474, LACZ vs. PRKACA p = 0.0002, LACZ vs. PIM1 p = 0.000007. For ZR-75-30, LACZ vs. PRKACA p = 0.0003, LACZ vs. PIM1 p = 0.0006.

PRKACA does not restore MAPK or AKT signaling, but does restore BAD phosphorylation. A. AEE788 or lapatinib treatment decreases p-ERK and p-AKT levels in HER2-amplified cells, and p-ERK levels are fully restored by activated HRAS (HRASV12) overexpression. B. PRKACA overexpression does not restore p-ERK or p-AKT levels in AEE788- or lapatinib-treated cells. For both panels, BT474 cells were transduced with the indicated pLX304 constructs and blasticidin selected. Lapatinib, AEE788, or vehicle control (DMSO) was added at the indicated concentrations, and cell lysates were harvested 24 h later for immunoblot analysis with the indicated antibodies. The V5 antibody identifies each of the V5-tagged ORFs, as indicated. C. Lapatinib or trastuzumab treatment results in loss of BAD phosphorylation, which is restored by PRKACA overexpression. BT474 cells were lentivirally-transduced with the indicated pLX304 constructs, blasticidin selected, and treated with lapatinib 0.1 uM or trastuzumab 10 ug/ml for 24 h. Protein lysates were harvested and subjected to immunoblot analysis with the indicated antibodies. The samples for the p-CREB and CREB immunoblots were run on the same gel, but the lapatinib and trastuzumab lanes were run in inverse orientation from the other panels, so the image was cut and re-aligned for labeling consistency with the other panels. D. BAD phosphorylation was restored by PRKACA overexpression in multiple HER2-positive cell lines after lapatinib treatment. SKBr3 and ZR-75-30 cells were lentivirally-transduced with pLX304 constructs expressing LACZ or PRKACA, blasticidin selected, and then treated with lapatinib 0.1 uM or DMSO for 24 h prior to immunoblot analysis with the indicated antibodies.

PRKACA is overexpresed in trastuzumab-resistant breast cancers. A. The kinase activity of PRKACA is required to confer resistance against lapatinib and for BAD phosphorylation. A closed (untagged) kinase-dead mutant of PRKACA (PRKACA-KD) was transduced into ZR-75-30 and SKBr3 cells alongside wild-type PRKACA constructs that were either V5-tagged or closed, as well as a LACZ control. Cells were treated with lapatinib at the indicated doses or DMSO and viability was assessed 6 days later by ATP-based luminescence assay. Results are normalized to the DMSO control for each ORF and represent the mean and SD of 3 replicates. B. Cells were lentivirally-transduced with pLX304-ORFs expressing V5-tagged PRKACA, closed PRKACA, closed PRKACA-KD, or LAZ. After blasticidin selection cells were treated with DMSO or lapatinib 0.1 uM for 24 h. Protein lysates were harvested and subjected to immunoblot analysis with the indicated antibodies. C. PRKACA is overexpressed in trastuzumab resistant human breast cancers. Immunohistochemical analysis for PRKACA expression in matched breast cancer samples from patients prior to any treatment (panels a, c, e) and after the development of resistance to trastuzumab-containing therapy (panels d, e, f). Magnification 40X.

PIM1 and BCL-XL confer resistance to anti-HER2 treatment in HER2-positive cells, and blockade of BCL-2/BCL-XL partially abrogates the resistance effect of PRKACA and PIM1. A. ZR-75-30 cells were transduced with the indicated pLX304 constructs. Two days later they were treated with increasing doses of lapatinib (left panel) or trastuzumab (right panel), and cell viability was assessed by ATP-based luminescence assay 6 days later. Results are normalized to the vehicle control for each ORF and represent the mean and SD of 3 replicates. B. BCL-XL overexpression confers resistance to lapatinib. ZR-75-30 cells were transduced with the indicated constructs. Two days later cells were treated with increasing doses of lapatinib, and cell viability was assessed by ATP-based luminescence assay 6 days later. Results are normalized to the vehicle control for each ORF and represent the mean and SD of 3 replicates. C. Immunoblot analysis of lysates from ZR-75-30 cells three days after ORF transduction in parallel with the cells used in (A) and (B) demonstrates exogenous expression of the indicated proteins. The anti-V5 antibody identifies each of the V5-tagged ORFs. D. PIM1 restores BAD ser112 phosphorylation after lapatinib treatment. ZR-75-30 cells were lentivirally-transduced with the indicated pLX304 constructs, blasticidin selected, and treated with DMSO or lapatinib 0.1 uM for 24h. Protein lysates were harvested and subjected for immunoblot analysis with the indicated antibodies. E. Pharmacological blockade of BCL-2 and BCL-XL synergizes with lapatinib treatment and partially abrogates the rescue effect conferred by PIM1 (top panel), PRKACA (middle panel), and BCL-XL (bottom panel). Two days after transduction with the indicated pLX304 constructs, ZR-75-30 cells were treated with the indicated doses of lapatinib and ABT-263 or DMSO. Cell viability was read out 6 days later by ATP-based luminescent assay. All results are normalized to the LACZ, DMSO-treated control and represent the mean and SD of 3 replicates. All three panels are from the same experiment using the same LACZ controls, but results are shown in separate graphs for ease of visualization. F. Immunoblot analysis of lysates from ZR-75-30 cells three days after ORF transduction in parallel with the cells used in (E) demonstrates exogenous expression of the indicated proteins. The anti-V5 antibody identifies each of the V5-tagged ORFs.